Method of regenerating bone/chondral tissues by transferring transcriptional factor gene

ABSTRACT

This invention provides a method of rapid and adequate culture of cells isolated from a body to effectively construct bone/cartilage tissues and implants containing the bone/cartilage tissues constructed by the aforementioned method. In this method, osteo-/chondro-inducible transcription factor genes are transfected into bone-marrow-derived cells isolated from a body using an adenoviral or a retroviral vector to grow on adequate scaffolds. The constructed bone/cartilage tissues are transplanted into a body together with the scaffolds. Thus, they can be used as substitutional bone/cartilage implants.

TECHNICAL FIELD

The present invention relates to a method for regeneration ofbone/cartilage tissues by transfection of osteo-/chondro-inducibletranscription factor genes. Particularly, the present invention relatesto a method of transfection of osteo-/chondro-inducible transcriptionfactor genes to accelerate cell differentiation into the target tissues,thereby effectively constructing bone/cartilage tissues andsubstitutional bone/cartilage implants produced by the aforementionedmethod.

BACKGROUND ART

Recently, in the field of regenerative medicine, in vitro culture andorganization of cells from a body have been attempted for reconstructingtissues that are as similar as possible to those in the body andreplacing the tissues in the body. In such tissue regeneration, theprovision of scaffolds for cell proliferation and acceleration of celldifferentiation are indispensable, along with the preservation of cells.

In general, cells exist in a body by adhering to the extracellularmatrix, which plays as a scaffold for differentiation and proliferation.Accordingly, suitable scaffolds for cell proliferation anddifferentiation are necessary in addition to cells, for in vitro cultureand complete three-dimensional tissue organization. Up to the present,the use of porous biodegradable materials such as porous ceramics asscaffolds has provided sufficient results in the regeneration of hardtissues such as bone and cartilage tissues.

In contrast, a method for rapid differentiation into target tissues invitro is important in the tissue engineering approach for tissueregeneration. Formally, however, cytokines (humoral factors) responsiblefor cell differentiation have been directly donated to cells or anexpression vector carrying cDNA of the target cytokine has beentransfected into cells by lipofection method and so on. A certain levelof success has been achieved by such methods.

Transfection of cell growth factors, however, did not provide sufficientresults by several problems. For example, the target cytokine did notalways fully and specifically affect the tissue, and the transfectionefficiency was almost 0 in primary cells although gene transfection bylipofection was somewhat successful in established cell lines.

Recently, many researchers have found that transcription factors,particularly transcription factors of runt and Helix-Loop-Helix (HLH)types, play significant roles in osteoblast differentiation. Forexample, a runt transcription factor, Pebp2alphaA (Pebp2αA)/Cbfa1, andan HLH transcription factor, such as Scleraxis, Id-1, and I-mfa, havebeen reported to have significant roles in osteoblast differentiation(e.g., Kunikazu Tsuji et al., “Runt transcription factor associated withosteoblast differentiation, Cbfa1/Pebp2αA, and Sox9 function inosteogenesis and chondrogenesis and unusual osteogenesis” ExperimentalMedicine, 16(11), 25-32, 1998). Special attempts in tissue regenerationutilizing functions of these transcription factors, however, have notyet been reported.

The present inventors have conducted concentrated studies. As a result,they have found that cells can be efficiently induced to differentiateinto bone/cartilage tissues by transfecting osteo-/chondro-inducibletranscription factors into bone-marrow-derived cells and having themexpress therein. Further, use of an adenoviral or a retroviral vectorfor the transfection of a transcription factor results in effectiveinfection to the primary adhesive cells such as bone/cartilage cells,and very effective induction of differentiation at approximately 99% canbe achieved. This has led to the completion of the present invention.

The present invention provides the following (1) to (13).

(1) A method for constructing bone/cartilage tissues, wherein anosteo-/chondro-inducible transcription factor gene is transfected intoisolated cells, and the isolated cells are allowed to differentiate andproliferate.

(2) The method according to (1) above, wherein the cells are derivedfrom bone marrow.

(3) The method according to (2) above, wherein the bone-marrow-derivedcells are mesenchymal stem cells.

(4) The method according to (2) above, wherein the bone-marrow-derivedcells are osteoblasts.

(5) The method according to any one of (1) to (4) above, wherein thecells are primary cells.

(6) The method according to (5) above, wherein the cells are isolatedfrom a patient.

(7) The method according to any one of (1) to (6) above, wherein theosteo-/chondro-inducible transcription factor is at least one memberselected from the group consisting of Cbfa1, Dlx-5, Bapx1, Msx2,Scleraxis, and Sox9.

(8) The method according to any one of (1) to (7) above, wherein theosteo-/chondro-inducible transcription factor gene is transfected intothe cells using an adenoviral or a retroviral vector.

(9) The method according to any one of (1) to (8) above, wherein thecells transfected by the osteo-/chondro-inducible transcription factorgenes proliferate in at least one scaffold selected from the groupconsisting of porous ceramics, collagen, polylactic acid, polyglycolicacid, and a complex thereof.

(10) A method for constructing bone/cartilage tissues comprising thefollowing procedures of:

-   -   1) inducing differentiation of bone-marrow-derived cells        isolated from a body with the aid of at least one member        selected from the group consisting of dexamethasone,        immunosuppressive factors, bone morphogenetic proteins, and        osteogenic cytokines;    -   2) transfection of the osteo-/chondro-inducible transcription        factor gene into the cells using an adenoviral or retroviral        vector; and    -   3) proliferation of the cells in at least one scaffold selected        from the group consisting of porous ceramics, collagen,        polylactic acid, polyglycolic acid, and a complex thereof.

(11) Implants containing the bone/cartilage tissues constructed by themethod according to any one of (1) to (10) above.

(12) The implants according to (11) above, which further contain atleast one scaffold selected from the group consisting of porousceramics, collagen, polylactic acid, polyglycolic acid, and a complexthereof.

(13) Implants containing cells, in which the osteo-/chondro-inducibletranscription factor gene is transfected, and at least one memberselected from the group consisting of porous ceramics, collagen,polylactic acid, polyglycolic acid, and a complex thereof.

DISCLOSURE OF THE INVENTION

The present invention is hereafter described in detail.

1. Construction of Bone/Cartilage Tissues

The present invention relates to a method of transfection of theosteo-/chondro-inducible (osteo-inducible and/or chondro-inducible)transcription factor gene to isolated cells to construct bone/cartilage(bone and/or cartilage) tissues in vitro by inducing differentiation andproliferation of the cells.

1.1 Transcription Factor

The transcription factors employed in the present invention are theosteo-/chondro-inducible transcription factors that induce immaturedcells to differentiate into bone and/or cartilage cells. Examplesthereof include Cbfa1, Dlx-5, Bapx1, Msx2, Scleraxis, and Sox9. Cbfa1 isa transcription factor that was cloned by Ogawa et al. at KyotoUniversity in 1993 and proved to be indispensable for inducingdifferentiation of the mesenchymal stem cell into the osteoblast byKomori et al. at Osaka University (Komori, T. et al., (1997) Cell 89,755-764). This Cbfa1 has two isoforms, til-1 and pebp2αA. Dlx-5 is ahomolog of the Drosophila distalless (Dll) gene and a transcriptionfactor associated with the ossification of perichondrium and endosporium(Acampora, D. et al., 1999, Development 126, 3795-3809). Bapx1 is ahomolog of the Drosophila bagpipe homeobox gene and is associatedparticularly with differentiation of the mesenchymal stem cell intocartilage cells in the spinal cord and is considered to be a regulatorygene of the Cbfa1 gene (Tribioli, C. et al., 1999, Development 126,5699-5711). Msx2 is a homolog of the Drosophila muscle segment homeobox(Msh) gene. It is associated with the ossification of calvaria and isconsidered to be a regulatory gene of the Cbfa1 gene (Satokata, I. etal., 2000, Nature Genet. 24, 391-395). Scleraxis is a transcriptionfactor that is associated with induction of differentiation from themesenchymal stem cell to cartilage cell or connective tissue (Cserjesi,P. et al., 1995, Development 121, 1099-1110). Sox9 is expressed incartilage and it regulates expression of the genes associated withcartilage differentiation of, for example, type II collagen (Ng, L. J.et al., 1997, Dev. Biol. 183, 108-121).

In the present invention, the above described osteo-/chondro-inducibletranscription factor genes can be prepared by conventional techniques,using their known sequences. For example, cDNA of a target transcriptionfactor can be prepared by extracting RNA from the osteoblast, preparingprimers based on a known sequence of the transcription factor gene, andcloning the cDNA by PCR using the RNA and the primers.

1.2 Induction of Cell Differentiation

The cells that are employed in the present invention are immatured cellswith differentiation and proliferation ability isolated from a body.Examples thereof include the ES (embryonic stem) cell, the mesenchymalstem cell, and the preosteoblast, differentiated from the mesenchymalstem cell. A bone-marrow-derived mesenchymal stem cell is particularlypreferable, and the osteoblast is more preferable. Particularly, whenthe present invention is applied in the field of regenerative medicine,the above-described cell is isolated from the body of a patient. Thiscell is preferably prepared by removing connective tissues with aconventional technique. Alternatively, cells may grow by primary culturebefore use.

The cells should be cultured in an appropriate medium for inducing celldifferentiation, constructing target tissues. In the case of bone tissueregeneration, for example, at least one member selected from the groupconsisting of dexamethasone, immunosuppressive factors such as FK-506and cyclosporine, bone morphogenetic proteins (BMP) such as BMP-2,BMP-4, BMP-5, BMP-6, BMP-7, and BMP-9, and osteogenic humoral factorssuch as TGFβ is preferably supplemented to the medium to induce celldifferentiation into bone cells.

1.3 Transfection of Transcription Factor Gene

In the present invention, the osteo-/chondro-inducible transcriptionfactor gene can be transfected into the target cell by a method that iscommonly employed for transfection to animal cells. Examples of themethods that can be employed include the calcium phosphate method,lipofection, electroporation, microinjection, and a method using anadenoviral, a retroviral, or a baculoviral vector. From the viewpointsof safety and transfection efficiency, a method using an adenoviral or aretroviral vector is preferable, and a method using an adenoviral vectoris most preferable.

The above-mentioned adenoviral or retroviral vector can be constructedaccording to a known technique. For example, an adenoviral vector can beconstructed according to the method of Miyake et al. (Miyake, S. et al.,Proc. Natl. Acad. Sci. 93: 1320-1324, 1993). Alternatively, acommercially available kit, such as the Adenovirus Cre/loxP Kit (TakaraShuzo Co., Ltd.), can be used. This kit is used for constructing arecombinant adenoviral vector utilizing a novel regulation system ofgene expression (Kanegae Y. et al., 1995, Nucl. Acids Res. 23, 3816)that employs Cre recombinase of P1 phage and its recognition sequence,loxP. This kit can be used to simply construct a recombinant adenoviralvector carrying the transcription factor gene. The multiplicity ofinfection (moi) of adenovirus infection is 10 or more, preferably 50 to200, and more preferably around 100 (approximately 80 to 120).

1.4 Cell Proliferation

The transfected cells should grow in suitable scaffolds in order toconstruct a complete three-dimensional structure. Examples of materialsthat can be used for scaffolds include hydroxyapatite or β-TCP(tricalcium phosphate), porous ceramics such as α-TCP, collagen,polylactic acid, polyglycolic acid, and a complex thereof (e.g., acomplex of polylactic acid, polyglycolic acid resin, and collagen).These scaffolds may be composed of a simple kind of material orcombinations of two or more kinds. Porous ceramics are particularlypreferable for scaffolds for tissue regeneration because of their highmechanical strength.

The above-mentioned scaffolds are preferably porous for uniforminoculation of cells. In the present description, the term “porous”refers to a porosity of 40% or higher. The pore size is not particularlylimited, although a diameter of 200 μm to 500 μm is preferable forfacilitated bone regeneration.

Cells can be proliferated by being inoculated and cultured in thescaffold in accordance with a conventional technique. Cells may besimply inoculated into the scaffold, or inoculated in the form ofmixtures with a liquid such as a buffer, physiological saline, a solventfor injection, or a collagen solution. When the cells do not smoothlyenter into a pore because of its porous structure of the material, cellsmay be inoculated under low pressure.

Preferably, the number of cells to be inoculated (inoculation density)is determined in accordance with the types of cells or scaffolds inorder to regenerate tissues more efficiently, maintaining the morphologyof the cells. For example, the inoculation density is preferably1,000,000 cells/ml or higher in the case of osteoblasts.

A conventional medium for cell culture, such as MEM medium, α-MEMmedium, or DMEM medium, can be suitably selected depending on the typeof cell to be cultured and then used for cell culture. FBS (Sigma) andantibiotics such as Antibiotic-Antimycotic (GIBCO BRL) or othersubstances may be added to the medium. Culture is preferably conductedin the presence of 3% to 10% CO₂ at 30° C. to 40° C., and particularlypreferably in the presence of 5% CO₂ at 37° C. The culture period is notparticularly limited, and it is at least 4 days, preferably at least 7days, and more preferably at least 2 weeks.

2. Implants Containing Bone/Cartilage Tissues

The bone/cartilage tissues constructed by the above mentioned method aretransplanted or injected into a body together with or separately fromthe scaffolds. Thus, they can be used as substitutional bone/cartilageimplants. Specifically, the present invention provides implantscontaining bone/cartilage tissues that are constructed in vitro.

Bone/cartilage tissues may be transplanted into the body separately fromscaffolds, although transplantation with scaffolds is preferable in thepresent invention. The most suitable scaffold for a given purpose andapplication site for implants may be selected from the above-mentionedscaffolds. For example, hydroxyapatite is preferable for atransplantation site (or a surgical technique) that requires mechanicalstrength. In contrast, biodegradable β-TCP or the like is preferable fora transplantation site (or a surgical technique) that does not requiremechanical strength.

The configurations and shapes of the implants of the present inventionare not particularly limited. Implants can take any desiredconfigurations or shapes, such as sponges, meshes, unwoven fabricproducts, discs, films, sticks, particles, or pastes. Theseconfigurations and shapes may be suitably selected depending on theirapplications.

The implants of the present invention can suitably contain othercomponents in addition to the bone/cartilage tissues and scaffoldswithin the scope of the present invention. Examples of such componentsinclude: growth factors such as basic fibroblast growth factors (bFGF),platelet-derived growth factors (PDGF), insulin and insulin-like growthfactors (IGF), hepatocyte growth factor, (HGF), glial-derivedneurotrophic factors (GDNF), neurotrophic factors (NF), hormones,cytokines, bone morphogenetic factors (BMP), transforming growth factors(TGF), and vascular endothelial growth factors (VEGF); bonemorphogenetic proteins; inorganic salts such as St, Mg, Ca, and CO₃;organic substances such as citric acid and phospholipid; and drugs.

Bone/cartilage tissues of the implants of the present invention areconstructed from the cells to which transcription factor genes have beentransfected. Bone/cartilage tissues may be constructed not only prior tothe transplantation (in vitro) but also after transplantation into bonedefects (in vivo). The implants of the present invention are highlycompatible with bones and of high bone formation ability. Thus, they canbe integrated with biological bones and can then restore bone defectsimmediately after they have been transplanted into a body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing constructions of type I Cbfa1 (pebp2αA) andtype II/III Cbfa1 (til-1).

FIG. 2 is a diagram showing a construction of pAxCALNLw cosmid for theCbfa1 gene transfection.

FIG. 3 is a photograph showing the results of X-gal staining for theevaluation of the LacZ expression level in rat osteoblasts.

FIG. 4 is a graph showing the results obtained by quantifying theexpression level of LacZ in rat osteoblasts.

FIG. 5 is a graph showing in the increase of alkaline phosphataseactivity in rat osteoblasts after the infection with recombinantadenovirus.

FIG. 6 is a graph showing in the increase of the calcium level in ratosteoblasts after the infection with recombinant adenovirus.

FIG. 7 is an image showing the results of expression level detectionregarding Cbfa1 genes by northern hybridization.

FIG. 8 is a graph showing the proliferation of rat osteoblasts after theinfection with recombinant adenovirus FIG. 9 is a photograph showing theresults of observation of calcification in rat osteoblasts after theinfection with recombinant adenovirus by Von Kossa staining.

FIG. 10 is a photograph showing the results of observation ofcalcification in rat osteoblasts after the infection with recombinantadenovirus by Alizarin Red staining.

FIG. 11 is a photograph showing the results of hematoxylin-eosinstaining of a tissue section obtained from the block 4 weeks aftertransplantation into rat's back subcutaneously.

FIG. 12 is a photograph showing the results of hematoxylin-eosinstaining of a tissue section obtained from the block 8 weeks aftertransplantation into rat's back subcutaneously.

FIG. 13 is a graph showing in the increase of alkaline phosphataseactivity in the block after transplantation into rat's backsubcutaneously.

FIG. 14 is a graph showing in the increase of the osteocalcin level inthe block after transplantation into rat's back subcutaneously.

FIG. 15 is a photograph showing the results of hematoxylin-eosinstaining of a tissue section obtained from the block aftertransplantation into rat's back subcutaneously and femur, wherein A-arepresents conditions 3 weeks after transplantation of the control intothe back; A-b represents conditions 5 weeks after transplantation of thecontrol into the back; A-c represents conditions 3 weeks aftertransplantation of the Cbfa1 (til-1)-infected cells into the back; A-drepresents conditions 5 weeks after transplantation of the Cbfa1(til-1)-infected cells into the back; B-a represents conditions 3 weeksafter transplantation of the control into the femur; B-b representsconditions 8 weeks after transplantation of the control into the femur;B-c represents conditions 3 weeks after transplantation of the Cbfa1(til-1)-infected cells into the femur; and B-d represents conditions 8weeks after transplantation of the Cbfa1 (til-1)-infected cells into thefemur.

FIG. 16 is a graph showing in the increase of alkaline phosphataseactivity in the block after it has been transplanted into rat's backsubcutaneously.

FIG. 17 is a graph showing in the increase of the osteocalcin level inthe block after transplantation into rat's back subcutaneously.

This description includes a part or all of the contents as disclosed inthe description of Japanese Patent Application No. 2001-227979, which isa priority document of the present application.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in more detail withreference to the examples, although the technical scope of the presentinvention is not limited thereto.

EXAMPLE 1 Bone Tissue Regeneration by Transfecting Cbfa1 Genes into RatOsteoblasts using an Adenoviral Vector

1. Experimental Method

1) Preparation of Adenoviral Vector

(i) Preparation of cDNA of Cbfa1

Two types of cDNAs of Cbfa1, i.e., type I: pebp2αA/Cbfa1 (SEQ ID NO: 1)and type II/III: til-1/Cbfa1 (SEQ ID NO: 2), were prepared from twotypes of plasmids*¹ (pSG5/IT (mPEBP2aA) and pSG5/KS (mtil-1)) providedby Sumitomo Pharmaceuticals Co., Ltd., ORF of mPEBP2aA was cleavedtherefrom using a BamHI restriction enzyme, and ORF of mtil-1 wascleaved therefrom using a BglII restriction enzyme (FIG. 1).*1: Two types of Cbfa1 exist, i.e., type I (pebp2αA/Cbfa1) expressedspecifically in T-cells and type II/III (til-1/Cbfa1) expressedspecifically in osteoblasts. Type II/III is longer than type I.

(ii) Preparation of recombinant Adenovirus

Two types of cDNAs of Cbfa1 were inserted into the SwaI site of thecosmid vector pAxCALNLw using the commercially available AdenovirusCre/loxP Kit (Takara Shuzo Co., Ltd.), and a recombinant adenoviralvector was prepared according to the manufacturer's instructionsattached to the kit (FIG. 2). The titer of the prepared virus wasapproximately 10¹¹ PFU/ml, and infection efficiency was very high.

2) Sampling and Culture of Bone Marrow Cells

The rat bone marrow osteoblast (RBMO) was sampled from the back femur ofa 6-week-old Fisher rat (male) in accordance with the method ofManiatopoulos et al. (Maniatopoulos, C., Sodek, J., and Melcher, A. H.,1988, Cell Tissue Res. 254, 317-330).

The sampled cells were cultured and in MEM medium (Nacalai Tesque)containing 15% FBS (Sigma) and Antibiotic-Antimycotic (GIBCO BRL) untilthey became confluent. Subsequently, cells were subcultured into theabove mentioned medium containing 5 nM dexamethasone (Sigma), 10 mMβ-glycerophosphate (Sigma), and 50 μg/ml ascorbic acid phosphate (Wako)in φ3.5 cm dishes, and they were cultured until the number of cells perdish became approximately 400,000. The subcultured rat osteoblast wasinfected with the recombinant adenoviral vector prepared as mentionedabove at a multiplicity of infection (moi) of 500 on the next day.Uninfected cells or cells infected with the mock vector were prepared asa control.

3) Observation of LacZ Gene-Expressing Cell by X-gal Staining

Expression of LacZ in the rat osteoblasts 3 days to 2 weeks afteradenovirus infection were observed by X-gal staining in accordance withthe method of Scholer et al. (Scholer, H. R. et al., 1989, EMBO J., 8,2551-2557) (FIG. 3). Further, the stained cells were subjected to imageanalysis using the NIH image, and the number of expressed cells wasdetermined to evaluate the efficiency of gene transfection (FIG. 4).

4) Assay of Alkaline Phosphatase Activity

The rat osteoblasts 3 days to 2 weeks after adenovirus infection werewashed with 100 mM Tris (pH 7.5) and 5 mM MgCl₂, collected using ascraper, suspended in 500 μl of 100 mM Tris (pH 7.5), 5 mM MgCl₂, and 1%Triton X-100, and then disrupted by sonication. After the sonication,cells were centrifuged at 6,000 g for 5 minutes to recover thesupernatant. The supernatant (5 μl in each case) was added to 0.056M2-amino-2-methyl-1,3-propanediol (pH 9.9), 10 mM p-nitrophenylphosphate, and 2 mM MgCl₂, the resultants were incubated at 37° C. for30 minutes, and then absorbance at 405 nm was measured using amicroplate reader immediately thereafter. The enzyme activity wasdetermined based on the calibration curve prepared using p-nitrophenolfrom the obtained absorbance (FIG. 5).

5) Assay of the Calcium Level

The rat osteoblasts 1 to 3 weeks after adenovirus infection were fixedwith 3.7% formalin buffer and then decalcified with 0.6M HCl all day andnight. The decalcified solution was diluted, and the calcium level wasassayed using a commercially available o-cresol phthaleincomplexon-containing reagent for calcium analysis (#587, 360-11, Sigma)according to the manufacturer's instructions (FIG. 6).

6) Northern Hybridization

Total RNA was extracted from the rat osteoblasts after adenovirusinfection using a commercially available TRIzol reagent (#15596-10551,GIBCO BRL) according to the manufacturer's instructions. Total RNA (10μg) was separated on a 1% agarose/5.5% formaldehyde gel and transferredin 20×SSC to the Hybond™-XL membrane (Amersham Pharmacia Biotech).Thereafter, the membrane was heated at 80° C. for 2 hours and theirradiated with ultraviolet rays for 2 minutes. The cDNA probes of GAPDHand Cbfa1 were labeled with α-³²PdCTP (3,000 Ci/mmol, Amersham PharmaciaBiotech) using Rediprime™ (Amersham Pharmacia Biotech), and theα-³²PdCTP that had not been incorporated was removed using a MicroSpin™G-25 Column (Amersham Pharmacia Biotech). This membrane was incubated inthe PerfectHyb™ Plus Hybridization Buffer (Sigma) at 68° C. for 30minutes, the labeled cDNA probe (2×10⁶ cpm/ml) was added, and incubationwas further carried out at 68° C. for 1 hour. The membrane was washedwith 2×SSC/0.11% SDS at room temperature for 5 minutes and then washedtwice with 0.5×SSC/0.1% SDS at 68° C. (20 minutes each). Thereafter, themembrane was exposed to the Kodak XAR film at −80° C. overnight (FIG.7).

7) Measurement of the Number of Cells

The osteoblasts 3 days to 2 weeks after adenovirus infection wereimmobilized with PBS containing 1 % glutaraldehyde for 5 minutes, washedtwice with distilled water, and then stained with 1% crystal violet atroom temperature for 30 minutes. After an excess amount of dye wasremoved by washing three times with distilled water, decolorization wascarried out with 10% acetic acid and 1% Triton X-100. The destainingsolution was diluted, and absorbance at 595 nm was measured. The numberof cells was determined based on the calibration curve*² from theobtained absorbance (FIG. 8).*2: The calibration curve was prepared by inoculating cells at anadequate density (duplicate), processing the cells by the above stainingtechnique, and counting the number of tripsinized cells.

8) Observation of Calcification

The conditions of calcification caused by mineral components secretedfrom the osteoblasts were observed by Von Kossa staining and AlizarinRed staining.

(i) Von Kossa Staining

The osteoblasts 1 to 3 weeks after adenovirus infection were fixed with3.7% formalin buffer for 5 minutes and then rinsed with diluted water.An aqueous solution of 5% silver nitrate was added, and incubation wasthen carried out for 20 minutes in the dark. Thereafter, incubation wasfurther carried out for 15 minutes in the light, and the resultant waswashed thoroughly with distilled water. The results were scanned in ascanner and then compared (FIG. 9).

(ii) Alizarin Red Staining

The osteoblasts 1 to 3 weeks after infection were fixed with 3.7%formalin buffer for 5 minutes and then rinsed with diluted water. Anaqueous solution of 1% Alizarin Red was added thereto, and incubationwas then carried out for 2 minutes. Thereafter, the resultant was washedthoroughly with distilled water. The results were scanned in a scannerand then compared (FIG. 10).

2. Results of Experimentation

1) Expression of lacZ Gene

As is apparent from FIG. 3 and FIG. 4, the expression level of the LacZgenes reached its peak 3 days after infection, and the LacZ genes wereexpressed in approximately 90% of cells. Thereafter, the number ofLacZ-expressing cells gradually decreased, although the expression levelwas maintained in some of the cells (approximately 20%) even 2 weeksafter infection.

2) Alkaline Phosphatase Activity

As is apparent from FIG. 5, the alkaline phosphatase activity of thecell in which Cbfa1 (pebp2αA, til-1) had been excessively expressed washigher than that of the control. Particularly, the enzyme activity 10days after infection became as high as approximately 6 times that of thecontrol.

3) Calcium Level

As is apparent from FIG. 6, the amount of calcium deposited in the cellin which Cbfa1 (pebp2αA, til-1) overexpressed cell was considerablylarger than that in the control.

4) Expression of Cbfa1 Genes

As is apparent from FIG. 7, a very high level of Cbfa1 gene expressionwas observed in the cell 3 days after the Cbfa1 (pebp2αA, til-1)recombinant adenovirus infection. Expression of endogenous Cbfa1 geneswas observed in the til-1-infected cell.

5) Cell Proliferation

As is apparent from FIG. 8, the virus titer of the Cbfa1 and Crerecombinant adenovirus infected cell was twice compared to that of theuninfected cell(control). There was no substantial difference in cellproliferation.

6) Calcification

As is apparent from the results of Von Kossa staining (FIG. 9) andAlizarin Red staining (FIG. 10), the level of calcification was moreadvanced in the cell in which Cbfa1 (pebp2αA, til-1) overexpressed cell.

3. Conclusion

Accordingly, cDNA of Cbfa1 was very effectively transfected into theosteoblasts with the aid of an adenoviral vector, and the transfectedCbfa1 gene would significantly accelerate the osteoblast differentiationinto bone tissues. This culture system was also found to be capable ofproviding effective ways for in vitro tissue regeneration through theuse of adequate scaffolds such as porous ceramics.

EXAMPLE 2 Experimentation for Transplanting the Cbfa1 Gene-TransfectedOsteoblasts into a Rat's Back Subcutaneously

1. Experimental Method

1) Transplantation into rat's Back Subcutaneously

The Cbfa1 gene-transfected rat osteoblasts were cultured using acommercially available β-TCP (tricalcium phosphate) porous block(average pore size: 200 μm in diameter, 5 mm×5 mm×5 mm, Olympus) as ascaffold in the following manner, and the composite was transplantedsubcutaneously to a rat's back. The confluent rat osteoblasts weretreated with trypsin and the cell suspension was then soaked into theβ-TCP block under low pressure (100 mHg) at a cell density of1,000,000/ml. After the cells were cultured for an additional 2 weeks,they were infected with the Cbfa1 (til-1)-recombinant adenoviral vectorprepared in Example 1 at an moi of 500, and the infected cells weretransplanted subcutaneously to a rat's back on the next day. As thecontrol, blocks comprising cells infected with the mock adenoviralvector were similarly transplanted. The blocks were excised 2, 4, and 8weeks after transplantation and then subjected to a variety of assaysand observations.

2) Observation of Tissue Sections (Hematoxylin-Eosin Staining)

The excised blocks were fixed with 4% paraformaldehyde and 0.05%glutaraldehyde by microwave radiation. On the next day, the resultantwas subjected to decalcification in 10% EDTA and 100 mM Tris (pH 7.4),and decalcification was continued for about 1 week. After thedecalcification, the blocks were dehydrated in ethanol and then embeddedin paraffin. Sctions prepared to a thickness of 5 μm each weredeparaffinized and stained with hematoxylin and eosin (FIGS. 11 and 12).

3) Assay of Alkaline Phosphatase Activity

The alkaline phosphatase activities of the excised blocks were assayedin the same manner as in Example 1 (FIG. 13).

4) Assay of the Osteocalcin Level

After the assay of alkaline phosphatase activity, the sediments of theblocks were disrupted by sonication in 20% formic acid, and incubated at4° C. for 4 days. After desalting with the use of a PD-10 column(Amersham Pharmacia Biotech), osteocalcin was extracted with 10% EDTAand 100 mM Tris (pH 7.4) and assayed the osteocalcin level using the RatOsteocalcin EIA Kit (Biomedical Technologies Inc.) (FIG. 14).

2. Result of Experimentation

1) Hematoxylin-Eosin staining

According to the results of hematoxylin-eosin staining of thetransplanted blocks (FIGS. 11 and 12), the number of cells existing inpores of the blocks was larger and the size of ossification area waslarger in the Cbfa1 (til-1) overexpressed cell.

2) Alkaline Phosphatase Activity

As is apparent from FIG. 13, the alkaline phosphatase activity of thecell in which Cbfa1 (til-1) overexpressed cell was higher than that ofthe control. Particularly, the enzyme activity 4 weeks aftertransplantation became as high as approximately 3 times that of thecontrol.

3) Osteocalcin Level

As is apparent from FIG. 14, the osteocalcin level in the Cbfa1 (til-1)overexpressed cell was higher than that in the control. Particularly,the osteocalcin level 4 weeks after transplantation became as high asapproximately 2.5 times that of the control.

EXAMPLE 3 Experimentation for Transplanting Cbfa1 Gene-TransfectedOsteoblasts to Rat's Back and Bone Defects

1. Experimental Method

1) Transplantation into Rat's back and into Femur

The rat osteoblasts were infected with the Cbfa1 (til-1)-recombinantadenovirus vector at a moi of 500. Twenty four hours later, the cellswere trypsinized and the cell suspension was soaked into thecommercially available β-TCP block (average pore size: 200 μm indiameter, 5 mm×5 mm×5 mm (for subcutaneous transplantation), 2 mm×2 mm×2mm (for bone defects transplantation), Olympus) at a cell density of2,000,000 cells/ml. Next day, the blocks were transplanted to a rat'sback and into the bone defects. Transplantation into the bone defectswas conducted by producing holes (diameter: 2 mm, depth: 3 to 4 mm) inthe frontal ends of left and right femora and by embedding the blocks inthe holes (bone defects). As the control, blocks containing uninfectedcells were similarly transplanted.

The subcutaneously transplanted block was excised 3 or 5 weeks aftertransplantation, the alkaline phosphatase activity and the osteocalcinlevel were assayed, and tissue sections were then observed. The blockthat had been transplanted into the femur was excised 3 or 8 weeks aftertransplantation, and tissue sections were then observed.

2. Result of Experimentation

1) Hematoxylin-Eosin Staining

Subcutaneous Transplantation:

The number of cells existing in pores of the blocks with the Cbfa1(til-1) overexpressed cell was larger, and bone formation was moreadvanced than that in the control, both 3 and 5 weeks aftertransplantation (FIG. 15A).

Transplantation into Bone Defects:

3 weeks after transplantation, bone formation was advanced, and thedissected area of the femur was almost completely replaced with new bonetissue in the block with the Cbfa1 (til-1) overexpressed cell. Incontrast, bone formation did not substantially occur in the case of thecontrol. In the block with the Cbfa1 (til-1) overexpressed cell, boneformation was further advanced, and the bone defect was almostcompletely replaced with bone marrow tissues 8 weeks aftertransplantation. In contrast, the transplanted block still remained asit had been in the control (FIG. 15B).

2) Alkaline Phosphatase Activity and Osteocalcin Level

The alkaline phosphatase activity in the block with the Cbfa1 (til-1)overexpressed cell was approximately 2.0 times and approximately 4.7times higher than that of the control 3 and 5 weeks aftertransplantation, respectively (FIG. 16). The osteocalcin level in thecell in which Cbfa1 (til-1) had been excessively expressed wasapproximately 2.0 times and approximately 1.5 times higher than that ofthe control 3 and 5 weeks after transplantation, respectively (FIG. 17).

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

Industrial Applicability

The present invention provides a method for effective in vitro tissueculture for bone/cartilage tissue regeneration using bone marrow cells.This method enables ultimate therapy for damaged tissues that cannot beregenerated in vivo, wherein a patient's own cells extracted fromhis/her body are cultured and organized in vitro to reconstruct tissuesthat are as similar as possible to those in the body, and the compositeis returned to the body.

1 A method for constructing bone/cartilage tissues, wherein anosteo-/chondro-inducible transcription factor gene is transfected intoisolated cells, and the isolated cells are allowed to differentiate andproliferate. 2 The method according to claim 1, wherein the cells arederived from bone marrow. 3 The method according to claim 2, wherein thebone-marrow-derived cells are mesenchymal stem cells. 4 The methodaccording to claim 2, wherein the bone-marrow-derived cells areosteoblasts. 5 The method according to any one of claims 1 to 4, whereinthe cells are primary cells. 6 The method according to claim 5, whereinthe cells are isolated from a patient. 7 The method according to any oneof claims 1 to 4, wherein the osteo-/chondro-inducible transcriptionfactor is at least one member selected from the group consisting ofCbfa1, Dlx-5, Bapx1, Msx2, Scleraxis, and Sox9. 8 The method accordingto any one of claims 1 to 4, wherein the osteo-/chondro-inducibletranscription factor gene is transfected into the cells using anadenoviral or a retroviral vector. 9 The method according to any one ofclaims 1 to 4, wherein the cells transfected by theosteo-/chondro-inducible transcription factor genes proliferate in atleast one scaffold selected from the group consisting of porousceramics, collagen, polylactic acid, polyglycolic acid, and a complexthereof. 10 A method for constructing bone/cartilage tissues comprisingthe following procedures of: 1) inducing differentiation ofbone-marrow-derived cells isolated from a body with the aid of at leastone member selected from the group consisting of dexamethasone,immunosuppressive factors, bone morphogenetic proteins, and osteogeniccytokines; 2) transfection of the osteo-/chondro-inducible transcriptionfactor gene into the cells using an adenoviral or retroviral vector; and3) proliferation of the cells in at least one scaffold selected from thegroup consisting of porous ceramics, collagen, polylactic acid,polyglycolic acid, and a complex thereof. 11 Implants containing thebone/cartilage tissues constructed by the method according to any one ofclaims 1 to
 4. 12 The implants according to claim 11, which furthercontain at least one scaffold selected from the group consisting ofporous ceramics, collagen, polylactic acid, polyglycolic acid, and acomplex thereof. 13 Implants containing cells, in which theosteo-/chondro-inducible transcription factor gene is transfected, andat least one member selected from the group consisting of porousceramics, collagen, polylactic acid, polyglycolic acid, and a complexthereof.